1. Field of the Invention
The present invention relates to prion isomers, referred to herein as PrPI. In another aspect, the present invention relates to methods of making prion isomers. In even another aspect, the present invention relates to compositions comprising a prion isomer peptide, and to methods of making said compositions. In still another aspect, the present invention relates to antibodies having specificity to at least one prion isomer, and to methods of making said antibodies. In yet another aspect, the present invention relates to products useful for assaying for the presence of prion isomers in a patient, and to methods of using said products. In even still another aspect, the present invention is directed to methods for treating a patient afflicted with a prion-associated disorder.
2. Description of the Related Art
Prions are infectious pathogens distinct from bacteria, viruses and viroids, and cause central nervous system spongiform encephalopathies in humans and animals. Examples of prion diseases include the mad cow disease, scrapie in sheep, and the human diseases Creutzfledt-Jacob disease, Gerstmann-Straussler-Scheinker disease, familial insomnia, and kuru (Gajdusek, 1977; Prusiner, 1997 and 1999; Weissmann and Aguzzi, 1997; Hill et al., 1997; Will et al., 1999). Thus, prion diseases are neuro-degenerative diseases that afflict both humans and animals.
Research indicates that prion diseases develop by a “protein only” mechanism (Griffith, 1967; Prusiner, 1982; Cohen and Prusiner, 1998; Balter,1999). The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. The underlying cause of prion disease is the conversion of a host derived cellular prion (PrPC) into an infectious scrapie prion (PrPSC) (Prusiner 1997 and 1999; Cohen and Prusiner, 1998; Horiuchi and Caughey, 1999). Interestingly, the PrPC and PrPSC proteins have the same molecular weight and amino acid sequences (Stahl et al., 1993) and differ only in their three-dimensional conformations.
The conformational difference between the non-infectious PrPC protein and the infectious PrPSC protein is associated with considerable dissimilarity of the physicochemical properties of the proteins. Unlike the PrPC protein, which is soluble, susceptible to enzyme digestion and rich in α-helical structure, the PrPSC protein is highly insoluble, partially resistant to proteolytic digestion and possesses a high content of β-sheet structure (Prusiner, 1999; Cohen and Prusiner, 1998; Jackson et al., 1999).
The PrPC protein is a sialoglycoprotein encoded by a gene that is located on human chromosome 20 (Oesch, B. et al., Cell 40:735-746, (1985); Basler, K. et al., 46:417-428 (1986); Liao, Y. J. et al., Science 233:364-367 (1986); Meyer, R. K. et al., Proc. Natl. Acad. Sci. USA 83:2310-2314 (1986); Sparkes, R. S. et al., Proc. Natl. Acad. Sci. USA 83:7358-7362 (1986); Bendheim, P. E. et al. J. Infect. Dis. 158:1198-1208 (1988); Turk, E. et al. Eur. J. Biochem. 176:21-30 (1988)). The PrP gene is expressed in neural and non-neural tissues, the highest concentration of mRNA being in neurons (Chesebro, B. et al., Nature 315:331-333 (1985); Kretzschmar, H. A. et al., Am. J. Pathol. 122:1-5 (1986); Brown, H. R. et al., Acta Neuropathol. 80:1-6 (1990); Cashman, N. R. et al., Cell 61:185-192 (1990); Bendheim, P. E., Neurology 42:149-156 (1992)). The translation product of PrP gene consists of 253 amino acids in humans (Kretzschmar, H. A. et al., DNA 5:315-324 (1986); Pucket, C. et al., Am. J. Hum. 49:320-329 (1991)), 254 in hamster and mice, and 256 amino acids in sheep. The PrP protein undergoes several post-translational modifications.
To date, the majority of prion structural data has been obtained by analyzing the structure of PrPC. The three-dimensional structures of mouse PrPC proteins of various lengths were determined by NMR analyses (Riek et al., 1996 and 1997; James et al., 1997; Donne et al., 1997; Daggett, 1998). Such analyses revealed that the protein contains three well-resolved α-helices and a short two-stranded β-sheets. PrPC contains one disulfide bond, is structurally plastic and may exist in various conformations under different conditions (Prusiner et al., 1993; Cohen and Prusiner, 1998). Attempts at investigating the structure of PrPSC have, unfortunately, been hampered by its intractable solubility.
Numerous efforts have been aimed at searching for conditions that generate prion isomers with an increased content of β-sheet structure, as well as PrPSC characteristics, in hopes of obtaining models with which PrP pathogenicity can be studied.
A scrapie-like prion rich in β-sheet structure was found to exist as a PrPC unfolding intermediate at pH 4.0 in the presence of 3.5 M urea (Hornemann and Glockshuber, 1998). A PrPC unfolding intermediate is also observed at pH 3.6-4.0 under 1-2M GdmCl (Swietnicki et al., 1997, 1998 and 2000; Zhang et al., 1997). Additionally, reduction of the disulfide bond in the presence of dithiothreitol (100 mM) and 6M GdmCl is capable of converting PrPC to a structure that exhibits high β-sheet content, is prone to aggregation and resistant to proteinase K (Jackson et al., 1999). Synthetic fragments of PrPC have also been shown to adopt β-rich conformations and to form amyloid, but these synthetic PrPC fragments fail to cause disease in rodents (Gasset et al., 1992). Cell-free induction of protease resistant prion in the presence of preexisting PrPSC has also been demonstrated (Kocisko et al., 1994; Caughey et al., 1999).
In spite of advancements in the art, conditions that permit fractionation and isolation of stable prion isomers have not been demonstrated. Thus, there remains a need for stable PrPSC-like prion peptides that are conformational isomers of PrPC wherein the isomers are useful as models for better understanding protein folding, prion pathogenicity, and prion-associated neuro-degenerative disorders.
There is another need in the art for methods of making said novel conformational isoforms of the prion protein.
There is even another need in the art for compositions comprising a peptide of a novel prion isoform useful in understanding and treating prion-associated disorders, and for methods of making such compositions.
There is still another need in the art for antibodies specific to prion isoforms useful for screening and/or treating a patient afflicted with a prion-associated disorder, preferably a neuro-degenerative disorder, and for methods of making such antibodies.
There is yet another need in the art for compositions comprising antibodies specific to prion isoforms useful for treating a patient afflicted with a prion-associated disorder, preferably a neuro-degenerative disorder, and for methods of making such compositions.
There is even still another need in the art for products comprising an antibody having specificity to at least one prion isomer.
There is even yet another need in the art for methods of detecting prion isomers in a patient.
There is still even another need in the art for methods of treating a patient afflicted with a prion-associated disorder, preferably a neuro-degenerative disorder.
These and other needs will become apparent to those of skill in the art upon review of this specification, including its drawings, claims and appendix.